Microorganism resistant to threonine analogue and production of biotin

ABSTRACT

A microorganism resistant to a threonine analogue, which has a plasmid containing part or whole of a biotin operon; and a process for producing biotin, which comprises culturing a microorganism described above in a medium to produce and accumulate biotin in the medium, and collecting biotin.

FIELD OF THE INVENTION

The present invention relates to a novel microorganism resistant to athreonine analogue and a process for producing biotin using themicroorganism. The biotin obtainable by the invention can be used as araw material for medicaments or cosmetics, feed additives, etc.

BACKGROUND OF THE INVENTION

Biotin (vitamin H) is a kind of vitamin B and is related to fatty acidsynthesis or saccharide metabolism as a coenzyme of a carboxylase. About10 tons of biotin has been produced by chemical synthesis processesevery year for use as a raw material for medicaments or cosmetics, feedadditives, etc. However, because these processes are complicated, biotinis very expensive. On the other hand, biotin production by fermentationprocesses has been studied for a long time. The fermentation processeshave not become practical because their productivity is low.

Biotin production using gene manipulation techniques has been expectedto provide inexpensive biotin. Some microorganisms modified by geneengineering techniques have been used for biotin production. Forexample, microorganisms belonging to the genus Escherichia such as astrain resistant to α-dehydrobiotin disclosed in e.g. JP-A 61-149091 areknown as the modified microorganisms for the biotin production. Otherknown modified microorganisms for the biotin production includemicroorganisms belonging to the genus Bacillus modified by transformingBacillus sphaericus and then providing resistance tothenoyltrifluoroacetone (JP-A 4-11894), microorganisms belonging to thegenus Serratia modified by providing Serratia marcescens SB411 withethionine-resistance followed by S-aminoethylcysteine-resistance andthen transforming the resulting microorganism with a recombinant plasmidcontaining a biotin gene fragment (JP-A 5-199867), transformants ofSerratia marcescens SB411 provided with resistance to actithiazic acid,a compound having biotin-like structure, or resistance to5-(2-thienyl)-n-valeric acid (JP-A 2-27980), and transformants providedwith resistance to a nicotinic acid analogue (Japanese PatentApplication No. 6-311778).

However, the prior art processes for producing biotin are unsatisfactoryfor the industrial production of biotin. There is still a need for aprocess for producing biotin having increased biotin productivity.

OBJECTS OF THE INVENTION

The main object of the present invention is to provide a microorganismwhich can produce biotin in high yield.

Another object of the present invention is to provide a process forproducing biotin using the above microorganism.

These objects as well as other objects and advantages of the presentinvention will become apparent to those skilled in the art from thefollowing description with reference to the accompanying drawings.

SUMMARY OF THE INVENTION

S-Adenosylmethionine is essential for the main synthetic pathway frompimelyl CoA to desthiobiotin (a biotin precursor). The present inventorshave expected that enhancing the biosynthetic pathway to methioninewould enhance the supplying system of S-adenosylmethionine, therebyimproving the accumulation of desthiobiotin and biotin.

In order to enhance the biosynthetic pathway to methionine, the presentinventors have isolated a strain resistant to a threonine analogue froma biotin-producing microorganism, and obtained a strain withsignificantly increased desthiobiotin and biotin accumulation. Afterfurther studies based on this finding, the present invention has beenaccomplished.

The present invention provides a microorganism resistant to a threonineanalogue, which has a plasmid containing part or whole of a biotinoperon.

The present invention also provides a process for producing biotin,which comprises culturing a microorganism described above in a medium toproduce and accumulate biotin in the medium, and collecting biotin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a regulatory region of the biotin operon and a basesequence near the bio B initiation codon.

FIG. 2 is a restriction map of DNA of plasmid pXBA 312.

DETAILED DESCRIPTION OF THE INVENTION

The threonine analogues include, for example, β-hydroxynorvaline.

The biotin operons include, for example, biotin operons derived frommicroorganisms belonging to the genera Escherichia, Bacillus, andSerratia. The biotin operons derived from microorganisms belonging tothe genus Escherichia include biotin operons derived from Escherichiacoli (JP-A 61-202686, etc.). The five genes bioA, bioB, bioF, bioC andbioD that are involved in biotin biosynthesis are encoded in the biotinoperon. In the invention, part of the biotin operon may be modified. Themodified biotin operons include, for example, those wherein at least onebase pair of either the base sequence of the regulatory region of thebiotin operon or the base sequence near the bioB initiation codon ofEscherichia coli is mutated as compared with the wild type. Theregulatory region of the biotin operon is the base sequence shown inSequence Listing 1 that is a base sequence of r-strand between bioA andbioB, specifically the base sequence of the region from bp (base pair)-1 to bp -86 shown in FIG. 1 when A of the bio B initiation codon ATG isconsidered bp 1. The base sequence near the bioB initiation codon is theregion from bp 1 to bp 6 when A of the bioB initiation codon ATG isconsidered bp 1. Specifically, at least one GC pair of upstream bp -53and bp -5 and downstream bp 4 when A of the bioB initiation codon ATG isconsidered bp 1 is mutated to an AT pair (JP-A 5-219956).

The plasmid to be used in the invention is a plasmid which can becarried by microorganisms belonging to the genus Escherichia, Bacillusor Serratia and the gene of which can be expressed. The plasmid ispreferably a plasmid carried by microorganisms belonging to the genusEscherichia. Examples of the plasmids include pXBA312 (derived fromEscherichia coli DRK-3323 (pXBA312)(FERM BP-2117), JP-A 2-502065),pXBRP319 (derived from Escherichia coli MM44/pXBRP319 (IFO 15721, FERMBP-4724), see Example 1 below), pAT 71 (derived from Escherichia coliHB/pAT71 (FERM BP-5668)), and derivatives thereof.

The microorganism of the invention can produce and accumulate biotin.Examples thereof include microorganisms belonging to the genusEscherichia, Bacillus or Serratia, etc. The microorganism is preferablya microorganism belonging to the genus Escherichia such as Escherichiacoli, preferably Escherichia coli HNV148/pXBRP319 (FERM BP-5667, IFO15894), Escherichia coli HB/pAT71 (FERM BP-5668, IFO 15895) obtained inExamples below, etc.

The microorganism resistant to a threonine analogue having a plasmidcontaining part or whole of the biotin operon is preferably amicroorganism resistant to a threonine analogue transformed with aplasmid containing part or whole of the biotin operon.

The microorganism of the invention can be obtained, for example, byproviding a parent strain of a microorganism with resistance to athreonine analogue and introducing a plasmid containing part or whole ofthe biotin operon into the resulting strain resistant to the threonineanalogue. The microorganism of the invention can also be obtained byintroducing a plasmid containing part or whole of the biotin operon intoa parent strain of a microorganism and providing the resultingmicroorganism with resistance to a threonine analogue. Alternatively,the microorganism can be obtained by providing with resistance to athreonine analogue a parent strain of a microorganism carrying a plasmidcontaining part or whole of the biotin operon.

As the parent strains, any microorganisms can be used in the inventionso long as they can produce and accumulate biotin. Examples of themicroorganisms include microorganisms belonging to the generaEscherichia, Bacillus and Serratia. The microorganisms belonging to thegenus Escherichia include, for example, microorganisms belonging toEscherichia coli, such as Escherichia coli IFO 14410, Escherichia coliW-3110 (IFO 12713) and its derivative strain Escherichia coli DR-85(JP-A 61-202686), Escherichia coli DR-332 (JP-A 62-155081), Escherichiacoli DRK-3323 (JP-A 2-502065), Escherichia coli BM4062 (JP-A 64-500081),Escherichia coli MS10/pXBRP319 (IFO 15570, FERM BP-4927) obtained inReference Examples below, and Escherichia coli ANA91/pXBRP319 (IFO15771, FERM BP-4928) obtained in Reference Examples below. The aboveEscherichia coli IFO 14410 and Escherichia coli IFO 12713 are knownstrains listed in List of Cultures, 9th edition (1992) (published byInstitute for Fermentation, Osaka, Japan (IFO)) and available from IFO.

The above Escherichia coli MS10/pXBRP319 has been deposited withNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology of 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan, under the Budapest Treaty under the accession umberof FERM BP-4927 since Dec. 12, 1994.

The above Escherichia coli ANA91/pXBRP319 has been deposited withNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology of 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan, under the Budapest Treaty under the accession umberof FERM BP-4928 since Dec. 12, 1994.

The above Escherichia coli HNV148/pXBRP319 has been deposited withNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology of 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan, under the Budapest Treaty under the accession umberof FERM BP-5667 since Oct. 30, 1995.

The above Escherichia coli HB/pAT71 has been deposited with NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology of 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan, under the Budapest Treaty under the accession umberof FERM BP-5668 since Oct. 30, 1995.

The microorganisms belonging to the genus Bacillus include, for example,microorganisms belonging to Bacillus sphaericus. Specific examplesthereof include Bacillus sphaericus IFO 3525 and its derivative strainBacillus sphaericus NZ-8802 (JP-A 4-11894). The above Bacillussphaericus IFO 3525 is a known strain listed in List of Cultures, 9thedition (1992) (published by IFO) and available from IFO.

The microorganisms belonging to the genus Serratia includemicroorganisms belonging to Serratia marcescens. Specific examplesthereof include Serratia marcescens Sn 41 and its derivative strainSerratia marcescens TA5024 (JP-A 2-27980), Serratia marcescens SB411 andits derivative strain Serratia marcescens ET2, Serratia marcescens ETA23(JP-A 5-199867), etc.

The above microorganisms can be used as such or as mutants thereof. Whenthe microorganisms contain no plasmid containing part or whole of thebiotin operon, if necessary, a plasmid containing part or whole of thebiotin operon is transformed into the microorganisms at a later step.

The strains resistant to a threonine analogue can be obtained by per seknown methods such as treatment with chemicals (e.g.,N-methyl-N'-nitro-N-nitrosoguanidine abbreviated as NTG), andultraviolet irradiation.

Then, a suspension of the resulting mutant cells is inoculated in aculture medium (e.g., agar plate culture medium) containing a threonineanalogue in an appropriate concentration, e.g., a concentration whichdoes not allow the growth of the parent strain. The grown colonies areisolated to conveniently obtain the strain resistant to the threonineanalogue.

The threonine analogue-resistant strain obtained in the above method iscultured, and the amount of biotin in the culture supernatant isdetermined to select microorganisms which can accumulate increasedamount of biotin.

The plasmid containing part or whole of the biotin operon can beintroduced into the microorganism by per se known methods. The plasmidcontaining part or whole of the biotin operon can be constructed by perse known methods, for example, by DNA cleavage with a restriction enzymefollowed by DNA linkage with T4DNA ligase (Molecular Cloning, Alaboratory Manual, Cold Spring Harbor Laboratory, 1982).

The host cell can be transformed with the above plasmid by per se knownmethods. For example, when the host is a bacterium belonging to thegenus Escherichia, the host can be transformed by the method describedin Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, 1982.

The microorganism resistant to a threonine analogue can be obtained bythe above method. Such microorganism can be used as it is. If necessary,such microorganism may further subjected to mutagenesis, and modifiedplasmids may be used.

The microorganism of the invention obtained in the above method iscultured in a medium to produce biotin in the medium.

The medium used for culture in the invention may be liquid or solid solong as it contains nutrition sources that the microorganisms to be usedcan utilize. For large scale culture, liquid media are preferably used.The medium contains assimilable carbon sources, assimilable nitrogensources, inorganic materials, trace nutrients, etc. The carbon sourcesinclude glucose, lactose, sucrose, maltose, dextrin, starch, mannitol,sorbitol, glycerol, fats and oils (e.g., soybean oil, olive oil, branoil, sesame oil, lard oil, chicken oil), and various fatty acids (e.g.,lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid).The nitrogen sources include meat extract, yeast extract, dried yeast,soybean flour, defatted soybean flour, corn steep liquor, peptone,cottonseed oil, blackstrap molasses, urea, thiourea, ammonia, andammonium salts (e.g., ammonium sulfate, ammonium chloride, ammoniumnitrate, ammonium acetate). In addition, salts including sodium,potassium, calcium, magnesium, etc., salts with metals such as iron,manganese, zinc, cobalt, nickel, etc., salts of inorganic acids such asphosphoric acid, boric acid, etc., salts of organic acids such as aceticacid, propionic acid, etc., can appropriately be used. In addition,amino acids (e.g., glutamic acid, aspartic acid, alanine, lysine,valine, methionine, proline), peptide (e.g., dipeptide, tripeptide),vitamins (e.g., vitamins B₁, B₂, B₁₂, C, nicotinic acid), nucleic acids(e.g., purine, pyrimidine and derivatives thereof), etc., can also beused. In order to control the pH in the medium, inorganic or organicacids, alkalis, etc., can be added. Appropriate amounts of oils andfats, surfactants, etc., can be used as antifoaming agents. The pH ofthe medium is preferably about 4 to 10, more preferably about 6 to 9.

The culture is any one of stationary culture, shaking culture, andaerobic and agitating culture. Aerobic and agitating culture ispreferred for large scale culture. The temperature for culture is 15 to42° C., preferably 30 to 37° C. The culture time varies with the cultureconditions, but is normally 1 to 10 days, preferably 2 to 4 days.

The culture is conducted by per se known methods such as batch culture,and fed-batch culture.

The resulting culture broth is centrifuged, and the amount of biotinaccumulated in the supernatant is determined by per se known methodssuch as bioassay using Lactobacillus plantarum as a test microorganism(The Vitamins, vol. 7, p. 303 (1967); Vitamins, Experimental Procedures(II), p. 475, edited by Japan Vitamin Society (1985); etc.).

The microorganism is cultured by the above method to produce andaccumulate biotin in the culture, and then biotin is collected from theculture. Because the biotin thus produced is present mainly in theculture filtrate, it is advantageous to separate the culture to obtainthe culture filtrate and cells by per se known methods (e.g.,filtration, centrifugation), and separate and purify biotin from theresulting filtrate. Alternatively, biotin can be purified directly fromthe culture broth.

The separation and purification can be carried out by per se knownmethods using difference in solubility in an appropriate solvent,precipitation from a solution, difference in precipitation rates,difference in various absorbance affinity, ion-exchange chromatographyusing ion-exchangers, concentration under reduced pressure,lyophilization, crystallization, recrystallization, drying, etc. Thesetechniques can be used alone or in an appropriate order of theircombination.

Biotin obtained by the invention can be used as raw materials formedicaments, cosmetics, etc., feed additives, etc.

The following reference examples and examples further illustrate theinvention in detail, but are not to be construed to limit the scope ofthe invention. All the percents (%) regarding the medium is W/V percents(W/V %).

Escherichia coli MS10/pXBRP319 and Escherichia coli ANA91/pXBRP319obtained in the following reference examples have been deposited atInstitute for Fermentation, Osaka, Japan (IFO) since Dec. 2, 1994 underthe Accession Numbers IFO 15770 and IFO 15771, respectively, and atNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology (NIBH), Tsukuba, Japan since Dec. 12,1994 under the Accession Numbers FERM BP-4927 and FERM BP-4928,respectively.

Escherichia coli HNV148/pXBRP319 and Escherichia coli HB/pAT71 obtainedin the following examples have been deposited at NIBH since Oct. 30,1995 under the Accession Numbers FERM BP-5667 and FERM BP-5668,respectively.

REFERENCE EXAMPLE 1

(1) Plasmid pXBA 312 (see FIG. 2) isolated from Escherichia coliDRK-3323/pXBA 312 (FERM BP-2117) (JP-A 2-502065) was cleaved with therestriction enzyme EcoRI, partially digested with PstI, and subjected toagarose gel electrophoresis and electroelution to isolate an EcoRI-PstIfragment (6.0 Kbp) containing the full-length biotin operon. Theresulting EcoRI-PstI fragment of pXBA 312 was ligated with an EcoRI-PstIfragment (3.6 Kbp) of plasmid pBR 322 to obtain plasmid pXBA 319.

Plasmid pMW 119 (Nippon Gene, Japan) was cleaved with the restrictionenzymes AatII and AvaI, and subjected to agarose gel electrophoresis andelectroelution to obtain an AatII-AvaI fragment (0.4 Kbp). Then, bothends of the AatII-AvaI fragment were made blunt ends with a blunting kit(Takara Shuzo Cc)., Ltd., Japan). The resulting fragment was ligated toSmaI site of pXBR 319 to obtain plasmid pXBRP 319.

(2) Plasmid pXBRP 319 obtained in above (1) was transformed into anexcellent strain obtained by mutagenesis treatment of Escherichia coliIFO 14410 (obtained from Institute for Fermentation, Osaka, Japan) withNTG. The resulting strain was further subjected to mutagenesis with NTGto isolate various drug-resistant strains. A strain producing a largeamount of biotin was selected from the drug-resistant strains to obtainEscherichia coli MM44/pXBRP 319 (FERM BP-4724).

(3) Escherichia coli MM44/pXBRP 319 obtained in above (2) was inoculatedin 2×YT medium (20 ml) containing yeast extract 10 g/L, peptone 16 g/Land sodium chloride 5 g/L, and subjected to shaking culture at 37° C.for 16 hours. The resulting culture solution (0.2 ml) was transferred to2×YT medium (20 ml), and subjected to shaking culture at 37° C. for 6hours. The resulting culture was centrifuged, and the collected cellswere rinsed twice with TM buffer (maleic acid 5.08 g/L, Tris 6.05 g/L,pH 6.0). The rinsed cells were suspended in TM buffer containing 200μg/ml of NTG, and subjected to mutagenesis at 37° C. for 25 minutes. Thetreated cells were collected by centrifugation and rinsed twice with TMbuffer, and suspended in the same buffer. The resulting suspension wasinoculated in an agar plate of M9 minimal medium containing 1 mg/mlβ-chloro-D-alanine, 4 μg/ml thiamine hydrochloride and 20 μg/ml Casaminoacid, and allowed to stand at 37° C. for 5 days to obtain colonies ofstrains resistant to β-chloro-D-alanine. One of the strains was selectedto obtain Escherichia coli BD10/pXBRP 319 (FERM BP-4725).

(4) Escherichia coli BD10/pXBRP 319 (FERM BP-4725) obtained in above (3)was subjected to mutagenesis with NTG, and various drug-resistantstrains were selected. A strain producing a large amount of biotin wasselected to obtain Escherichia coli MS10/pXBRP 319.

(5) Escherichia coli MS10/pXBRP 319 obtained in above (4) was inoculatedin 2×YT medium (20 ml) containing yeast extract 10 g/L, peptone 16 g/Land sodium chloride 5 g/L, and subjected to shaking culture at 37° C.for 16 hours. The resulting culture solution (0.2 ml) was transferred to2×YT medium (20 ml), and subjected to shaking culture at 37° C. for 6hours. The resulting culture broth was centrifuged, and the collectedcells were rinsed twice with TM buffer (maleic acid 5.08 g/L, Tris 6.05g/L, pH 6.0). The rinsed cells were suspended in TM buffer containing200 μg/ml of NTG, and subjected to mutagenesis at 37° C. for 25 minutes.The treated cells were collected by centrifugation and rinsed twice withTM buffer, and suspended in the same buffer. The resulting suspensionwas inoculated in an agar plate of M9 minimal medium containing 30 μg/ml6-aminonicotinamide, 4 μg/ml thiamine hydrochloride and 20 μg/mlCasamino acid, and allowed to stand at 37° C. for 5 days to obtaincolonies of strains resistant to 6-aminonicotinamide. One of the strainswas selected and was designated as Escherichia coli ANA91/pXBRP319 (FERMBP-4928).

REFERENCE EXAMPLE 2

Escherichia coli ANA91/pXBRP319 obtained in Reference Example 1 wasgrown at 37° C. for 16 hours in a 200 ml creased flask containing a seedmedium (pH 7.1, 30 ml) composed of glucose 2%, calcium carbonate 1%,corn steep liquor 4%, ammonium sulfate 0.4%, KH₂ PO₄ 0.1%, K₂ HPO₄ 0.2%and MgSO₄ •7H₂ O 0.01% on a rotary shaker. The resulting culture (0.6ml) was transferred to a 200 ml creased flask containing a main medium(pH 7.1, 30 ml) composed of glucose 5%, corn steep liquor 5%, ammoniumsulfate 0.2%, DL-alanine 0.3%, KH₂ PO₄ 0.1%, K₂ HPO₄ 0.2%, MgSO₄ •7H₂ O0.01%, FeSO₄ •7H₂ O 0.001% MnSO₄ •4-6H₂ O 0.001% and thiaminehydrochloride 0.002%, and grown at 37° C. for 30 hours on a rotaryshaker at 220 rpm. After completion of the cultivation, the culture wascentrifuged. The quantitative analysis of biotin in the culturesupernatant showed that 160 mg/ml biotin was accumulated.

EXAMPLE 1

Escherichia coli ANA91/pXBRP 319 obtained in Reference Example 1 wasinoculated in 2×YT medium (20 ml) containing yeast extract 10 g/L,peptone 16 g/L and sodium chloride 5 g/L, and subjected to shakingculture at 37° C. for 16 hours. The resulting culture solution (0.2 ml)was transferred to 2×YT medium (20 ml), and subjected to shaking cultureat 37° C. for 6 hours. The resulting culture broth was centrifuged, andthe collected cells were rinsed twice with TM buffer (maleic acid 5.08g/L, Tris 6.05 g/L, pH 6.0). The rinsed cells were suspended in TMbuffer containing 200 μg/ml of NTG, and subjected to mutagenesis at 37°C. for 25 minutes. The treated cells were collected by centrifugationand rinsed twice with TM buffer, and suspended in the same buffer. Theresulting suspension was inoculated in an agar plate of M9 minimalmedium containing 2 g/L β-hydroxynorvaline, and allowed to stand at 37°C. for 5 days to obtain colonies of strains resistant toβ-hydroxynorvaline. One of the strains was selected and was designatedas Escherichia coli HNV148/pXBRP319 (FERM BP-5667, IFO 15894).

EXAMPLE 2

Escherichia coli HNV148/pXBRP319 obtained in Example 1 was grown at 37°C. for 16 hours in a 200 ml creased flask containing a seed medium (pH7.1, 30 ml) composed of glucose 2%, calcium carbonate 1%, corn steepliquor 4%, ammonium sulfate 0.4%, KH₂ PO₄ 0.1%, K₂ HPO₄ 0.2% and MgSO₄•7H₂ O 0.01% on a rotary shaker at 220 rpm. The resulting culture (0.6ml) was transferred to a 200 ml creased flask containing a main medium(pH 7.1, 30 ml) composed of glucose 5%, corn steep liquor 5%, calciumcarbonate 2%, ammonium sulfate 0.2%, DL-alanine 0.3%, KH₂ PO₄ 0.1%, K₂HPO₄ 0.2%, MgSO₄ •7H₂ O 0.01%, FeSO₄ •7H₂ O 0.003%, MnSO₄ •4-6H₂ O0.003% and thiamine hydrochloride 0.002%, and grown at 37° C. for 30hours on a rotary shaker at 220 rpm. After completion of thecultivation, the culture was centrifuged. The biotin accumulation in theresulting culture supernatant was quantified by the bioassay usingLactobacillus plantarum IFO 3070 as a test microorganism, and found tobe 175 mg/L. In contrast, when the parent strain was cultured under thesame conditions, the biotin accumulation was only 160 mg/L.

EXAMPLE 3

Escherichia coli HNV148/pXBRP319 obtained in Example 1 was grown at 37°C. for 16 hours in a 500 ml creased flask containing a seed medium (pH7.1, 125 ml) composed of glucose 2%, calcium carbonate 1%, corn steepliquor 4%, ammonium sulfate 0.4%, KH₂ PO₄ 0.1%, K₂ HPO₄ 0.2%, MgSO₄ •7H₂O 0.01%, FeSO₄ •7H₂ O 0.05%, thiamine hydrochloride 0.002% andtetracycline hydrochloride 0.0012% on a rotary shaker at 210 rpm. Thetotal amount of the culture thus obtained was transferred to a 5 literjar fermentor containing a main medium (pH 7.1, 2.5 L) composed ofglucose 3%, corn steep liquor 6%, ammonium sulfate 0.2%, KH₂ PO₄ 0.1%,K₂ HPO₄ 0.2%, MgSO₄ •7H₂ O 0.02%, DL-alanine 0.3%, MnSO₄ •4-6H₂ O0.003%, FeSO₄ •7H₂ O 0.003%, Fe₂ (SO₄)₃ •nH₂ O 0.02%, thiaminehydrochloride 0.002%, 25% ammonia water 1.6 ml/L and Actocoal (anantifoaming agent manufactured by Takeda Chemical Industries, Ltd.)0.02%, and grown at 37° C. at an aeration rate of 2.5 L/minute. Theagitation speed was increased from 550 to 850 rpm in proportion to theamount of the cells. An aqueous solution of glucose (66.7%) wascontinuously added so that the glucose concentration was in the range of0.1 to 0.5%. During the cultivation, 25% ammonia water was added tomaintain the pH in the range of 6.5 to 7.0. If necessary, Actocoal wasadded for antifoaming. The cultivation for 72 hours gave a culturecontaining biotin (710 mg/L).

EXAMPLE 4

(1) Plasmid pAMP72 (JP-A 5-219956) was completely digested with therestriction enzymes NcoI and EcoT22I, and a 1 kb fragment containing abiotin operon promoter was isolated and recovered by agarose gelelectrophoresis. Meanwhile, pXBRP 319 was completely digested with NcoIand EcoT22I in the same manner, and an about 10 kb fragment wasrecovered. The about 10 kb fragment was ligated with the above 1 kbfragment to obtain plasmid pXBRP71.

(2) The plasmid pXBRP 71 obtained in above (1) was completely digestedwith the restriction enzymes EcoRI and SalI and subjected to agarose gelelectrophoresis to obtain a 7 kb fragment containing a biotin operon.

Meanwhile, plasmid pBR 322 (manufactured by Takara Shuzo Co., Ltd.Japan) was completely digested with the restriction enzyme AvaI,precipitated with ethanol, and made blunt ends with a blunting kit(manufactured by Takara Shuzo Co., Ltd. Japan). After inactivation withheat, the resulting fragment was completely digested with therestriction enzyme EcoRI, and separated by agarose gel electrophoresisto obtain a 1.4 kb fragment containing a tetracycline-resistant gene.Plasmid pSTV28 (manufactured by Takara Shuzo Co., Ltd. Japan) wascompletely digested with the restriction enzymes XmnI and SalI to obtaina 1.1 kb fragment containing a replication origin. The three fragmentsthus obtained were ligated to make plasmid pAT71.

(3) Escherichia coli HNV148/pXBRP319 was subcultured to obtainEscherichia coli HNV148 in which plasmid pXBRP319 had been removed. Theresulting strain HNV 148 was inoculated in an agar plate of M9 minimalmedium containing no thiamine, and allowed to stand at 37° C. for 4 daysto obtain thiamine--non-requisite strains. One of these strains wasselected and designated as Escherichia coli HB.

(4) Plasmid pAT71 obtained in above (2) was introduced into Escherichiacoli HB obtained in above (3) to obtain Escherichia coli HB/pAT71 (IFO15895).

EXAMPLE 5

Escherichia coli HB/pAT71 obtained in Example 4 was grown at 37° C. for16 hours in a 200 ml creased flask containing a seed medium (pH 7.1, 30ml) composed of glucose 2%, calcium carbonate 1%, corn steep liquor 4%,ammonium sulfate 0.4%, KH₂ PO₄ 0.1%, K₂ HPO₄ 0.2% and MgSO₄ •7H₂ O 0.01%on a rotary shaker at 220 rpm. The resulting culture (0.6 ml) wastransferred to a 200 ml creased flask containing a main medium (pH 7.1,30 ml) composed of glucose 5%, corn steep liquor 5%, calcium carbonate2%, ammonium sulfate 0.2%, DL-alanine 0.3%, KH₂ PO₄ 0.1%, K₂ HPO₄ 0.2%,MgSO₄ •7H₂ O 0.01%, FeSO₄ •7H₂ O 0.003%, MnSO₄ •4-6H₂ O 0.003% andthiamine hydrochloride 0.002%, and grown at 37° C. for 30 hours on arotary shaker at 220 rpm. After completion of the cultivation, theculture was centrifuged. The biotin accumulation in the resultingculture supernatant was quantified and found to be 200 mg/L.

EXAMPLE 6

Escherichia coli HB/pAT71 obtained in Example 4 was grown at 37° C. for16 hours in a 500 ml creased flask containing a seed medium (pH 7.1, 125ml) composed of glucose 2%, calcium carbonate 1%, corn steep liquor 4%,ammonium sulfate 0.4%, KH₂ PO₄ 0.1%, K₂ HPO₄ 0.2%, MgSO₄ •7H₂ 0 0.01%,FeSO₄ •7H₂ 0 0.05%, thiamine hydrochloride 0.002% and tetracyclinehydrochloride 0.0012% on a rotary shaker at 210 rpm. The total amount ofthe culture thus obtained was transferred to a 5 liter jar fermentorcontaining a main medium (pH 7.1, 2.5 L) composed of glucose 3%, cornsteep liquor 6%, ammonium sulfate 0.2%, KH₂ PO₄ 0.1%, K₂ HPO₄ 0.2%,MgSO₄ •7H₂ O 0.05%, DL-alanine 0.3%, MnSO₄ -4-6H₂ O 0.003%, FeSO₄ •7H₂ O0.003%, Fe₂ (SO₄)₃ •nH₂ O 0.1%, calcium citrate 1%, thiaminehydrochloride 0.002%, 25% ammonia water 1.6 ml/L and Actocoal (anantifoaming agent manufactured by Takeda Chemical Industries, Ltd.)0.02%, and grown at 37° C. at an aeration rate of 2.5 L/minute. Theagitation speed was increased from 550 to 950 rpm in proportion to theamount of the cells. An aqueous solution of glucose (66.7%) wascontinuously added so that the glucose concentration was in the range of0.1 to 0.5%. During the cultivation, 25% ammonia water and 30% potassiumhydroxide were added to maintain the pH in the range of 6.5 to 7.0. Ifnecessary, Actocoal was added for antifoaming. At 24 to 72 hours afterthe beginning of the culture, 1.25% ferric citrate (300 ml) was fed. Thecultivation for 82 hours in this manner gave a culture containing biotin(970 mg/L).

As described above, the microorganism of the invention has excellentbiotin-productivity. Culturing the microorganism can produce a largeamount of biotin.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  1                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  114 bas - #es                                                    (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE: Genomic DNA                                       - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM:  Escheric - #hia coli                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CGTCCGTTGT CATAATCGAC TTGTAAACCA AATTGAAAAG ATTTAGGTTT  - #                  50                                                                         - - ACAAGTCTAC ACCGAATTAA CAACAAAAAA CACGTTTTGG AGAAGCCCCA  - #                 100                                                                         - - TGGCTCACCG CCCA              - #                  - #                      - #    114                                                                 __________________________________________________________________________

We claim:
 1. A microorganism which is a recombinant Escherichia coli.,said recombinant being able to grow in a medium containingβ-hydroxynorvaline in such concentration that its parent strain cannotgrow and being capable of producing biotin, wherein said microorganismis Escherichia coli HNV148/pXBRP319 (FERM BP-5667).
 2. A microorganismwhich is a recombinant Escherichia coli., said recombinant being able togrow in a medium containing β-hydroxynorvaline in such concentrationthat its parent strain cannot grow and being capable of producingbiotin, wherein said microorganism is Escherichia coli HB/pAT71 (FERMBP-5668).
 3. A process for producing biotin, which comprises culturing amicroorganism according to claim 1 in a medium to produce and accumulatebiotin in the medium, and collecting the biotin.
 4. A process forproducing biotin, which comprises culturing a microorganism according toclaim 2 in a medium to produce and accumulate biotin in the medium, andcollecting the biotin.